Nucleic acid antioxidant compositions, methods for obtaining such compositions and formulations thereof

ABSTRACT

Compositions containing purified nucleic acid wherein the nucleic acid acts as an antioxidant. Such compositions also include materials subject to oxidative damage such as antioxidants, vitamins, lipids, foods and pharmaceuticals. The invention also includes methods for preparing such compositions. These methods include dissolving the nucleic acid and a hydrophilic material in an aqueous solution, which may later be dried or further processed. Additionally, nucleic acid may be coupled with a molecule having hydrophobic and hydrophilic regions and then solubilized in a hydrophobic material. It may also be shaped into small aggregates and added to a hydrophobic material.

FIELD OF THE INVENTION

The present invention relates to the use of a nucleic acid, such as DNAas an antioxidant. It includes nucleic acid antioxidant compositions andmethods of obtaining such compositions. It also includes formulations inwhich a nucleic acid is used as an antioxidant.

BACKGROUND OF THE INVENTION

Antioxidants are widely used to preserve a variety of materialsincluding food, vitamins, and pharmaceuticals. Such materials arenaturally degraded by oxidants, such as oxygen radicals, present in thematerial or its environment. These oxidants are highly reactive andaccordingly bond to and/or break down other chemicals in the material,resulting in the appearance of undesirable properties in the material.To avoid these undesirable effects, an antioxidant may be added to thematerial. The antioxidant has a strong affinity for oxidants andtherefore provides a preferred reactant. Some such reactions may producean undesirable by-product, but many do not. Antioxidants used forpreservative purposes are selected to avoid undesirable by-products.

Foods commonly treated with antioxidants include oils, which are oftenused for cooking and tend to oxidize more rapidly at highertemperatures, oil-containing foods such as coffee, and dried foods. Manyvitamins, including Vitamins C and E, are themselves antioxidants andtherefore are rapidly degraded unless another antioxidant is providedwith the vitamin. A large number of pharmaceuticals, such as syntheticestrogens, also benefit from the addition of antioxidants, especially ifoxidative damage may result in a toxic by-product.

In addition to their preservative effects, many antioxidants also havephysiological effects. Antioxidants are known to reduce the inflammatoryresponse and may be used in conjunction with other drugs to treatconditions resulting from or worsened by inflammation. Additionally, itis believed that ingestion of some antioxidants, such as Vitamins C andE, helps prevent cancer and other disorders caused by oxidative damageto the body.

Accordingly, many antioxidants are available at present. These includeartificial antioxidants such as butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA) and tert-butyl hydroquinone (TBHQ).Vitamin E, generally used to describe a class of chemicals known as“tocopherols”, and Vitamin C, also commonly called “ascorbic acid”, arealso used as antioxidants. Natural antioxidants such as tea extracts andextracts of herbs such as rosemary are also used for antioxidantpurposes.

SUMMARY OF THE INVENTION

The invention includes a method of preserving a material subject tooxidative damage by adding purified nucleic acid to the material. Thenucleic acid reduces the rate of oxidative damage of the material.

In specific embodiments of the present invention, the nucleic acid mayinclude DNA. The material subject to oxidative damage may be a vitaminsuch as Vitamin C or Vitamin E. The material subject to oxidative damagemay also be a food, and antioxidant, a lipid or a pharmaceutical.

In specific embodiments where the material subject to oxidative damageis hydrophilic, the purified nucleic acid may be in an aqueous solution.Where the material subject to oxidative damage is hydrophobic, thenucleic acid may be supplied in a solvent with both hydrophilic andhydrophobic regions or as aggregates of nucleic acid less than 50 μM indiameter.

In certain embodiments, the purified nucleic acid may be sprayed on thesurface of the material subject to oxidative damage.

The invention also includes a composition including a material subjectto oxidative damage and an amount of purified nucleic acid sufficient toreduce the rate of oxidative damage to the material.

In specific embodiments, the nucleic acid may include DNA. The materialsubject to oxidative damage may be a vitamin such as Vitamin C orVitamin E. The material subject to oxidative damage may also be a food,and antioxidant, a lipid, such as a lipid heated above ambienttemperature, or a pharmaceutical, such as a synthetic estrogen.

DETAILED DESCRIPTION

For a better understanding of the invention, reference may be had to thefollowing description of exemplary embodiments.

The present invention includes nucleic acid compositions in which thenucleic acid serves as an antioxidant. Although DNA may be best suitedfor most embodiments of the invention because of its durability, RNA orother nucleic acids may also be used, especially in applications wheremore rapid degradation of the nucleic acid is desirable. DNA and RNA mayinclude all forms of DNA and RNA, natural and synthetic, specificallyincluding cDNA, mRNA, rRNA and tRNA.

The nucleic acid compositions may be part of a material, such as a food,vitamin, cosmetic, or pharmaceutical. The nucleic acid compositions mayalso be formulated for later addition to such a material. Suchcompositions are biologically safe. Oxidized nucleic acid breaks downinto readily degradable and non-toxic by-products. Additionally, DNA isheat-tolerant; the base pairs do not normally separate in an aqueoussolution until approximately 94–96° C. and may remain bonded at highertemperatures in non-aqueous solutions. Nucleic acid antioxidants of thepresent invention may be provided and/or dispensed in powder liquid,gel, spray or aerosol forms, inter alia, or as a misting agent.

The nucleic acid used in the present invention may be from any source.Harvesting techniques for recovery of nucleic acid from biologicalsources, including techniques capable of producing commercial volumes ofnucleic acids are readily known in the art. Nucleic acids may beextracted from almost any biological source. Two common sources ofnon-specific nucleic acid are fish sperm and calf thymus. Almost anysource, animal or plant-based, yeast or bacterial may be used. Thesesources may be specifically developed for nucleic acid harvest or may bewaste products of other commercial processes, as in the case of calfthymus. Because the present invention employs nucleic acids for theirantioxidant properties alone, and not for their information codingproperties, the sequences of the nucleic acid may be irrelevant.

DNA, in certain examples, may be produced by solubilization of cellularmaterial with a detergent, followed by extraction of nucleic acid fromthe aqueous layer with an alcohol. Various additional steps andadditives may assist in the removal of protein to obtain purer nucleicacid. Various nucleases and extraction techniques may be employed todestroy unwanted forms of nucleic acids, such as RNA. Such techniquesare well known in the art.

Nucleic acids may also be synthesized artificially from nucleotides. Forinstance, surface catalysis techniques or oligonucleotide synthesizersmay be used.

The purity of nucleic acid from biological sources used in compositionsof the present invention may vary by application. In most applications,the nucleic acid will contain no more than 50% by weight residual matterfrom the biological source such as proteins, lipids and carbohydrates.In certain embodiments, it will contain no more than 25%, 10%, 5% or 1%by weight residual matter. Such residual matter will likely includeproteins which may cause unwanted effects such as bad taste in food orspoilage. For pharmaceutical compositions, the nucleic acid may containless than 5% by weight residual matter. Residual protein may need to besubstantially removed from pharmaceutical compositions, especiallyinjectable compositions, in order to avoid an immune response.

Nucleic acid is biodegradable and may also degrade due to oxidativedamage, which is known to cause breaks in nucleic acid molecules. Inmany applications the rate of degradation of nucleic acid will not besignificant. However, the rate may be influenced by the length of thenucleic acid molecule used and type of nucleic acid as well as bytreatment of the nucleic acid.

The nucleic acid may also be crosslinked, although such crosslinking mayreduce the antioxidant properties of the nucleic acid. Additionally, ifthe nucleic acid composition will eventually be used in a biologicalsystem, such as a human body, crosslinking agents that are toxic in sucha biological system may be avoided. Crosslinking may be between chainsof a single DNA molecule or between chains of two different nucleic acidmolecules or in any other possible permutation. Crosslinking may beaccomplished in a variety of ways, including hydrogen bonds, ionic andcovalent bonds, ππ bonds, van der Wals forces. More specifically,crosslinking may be accomplished by UV radiation, esterification,hydrolysis, or silica compounds if biological toxicity is at issue. Onespecific example includes the use of siloxane bridges as described inU.S. Pat. No. 5,214,134. Intercalating agents, neoplastic agents,formaldehyde and formalin may also be used.

More than one type of crosslinking may be used in a given composition.Furthermore, crosslinking may occur between two strands of a nucleicacid molecule or between two separate nucleic acid molecules. Increasedlevels of crosslinking will generally slow degradation of nucleic acid,but may result in lower antioxidant activity. However, in someapplications any reduced antioxidant effect may be worth the increasedstability. For example, although DNA is heat-resistant, basepair-crosslinked DNA will not be as able to separate along base pairs athigher temperatures and thus will exhibit at greater degradationresistance at higher temperatures. An optimal balance betweendegradation and antioxidant activity for a given composition should bereadily determinable to one skilled in the art.

Additionally, the nucleic acid may be methylated, ethylated, alkylated,or otherwise modified along the backbone to influence degradation rates.Generally, methylated, hemi-methylated, ethylated, or alkylated nucleicacids will degrade more slowly. Other backbone modifications affectingdegradation rates include the use of heteroatomic oligonucleosidelinkages as described in U.S. Pat. No. 5,677,437. Such backbonemodifications may also affect the solubility of nucleic acid, forinstance rendering it more lipid soluble. Backbone modifications mayalso increase the antioxidant capacity of nucleic acid.

Nucleic acids may also be capped to prevent degradation, influencesolubility, or influence antioxidant effects. Such caps are generallylocated at or near the termini of the nucleic acid chains. Examples ofcapping procedures are included in U.S. Pat. Nos. 5,245,022 and5,567,810.

The size of the nucleic acid molecules used in compositions of thepresent invention may vary from as small as 2 bases to as long as 10,000bases or more. In general, most compositions will contain nucleic acidmolecules with a variety of lengths. In exemplary embodiments, theaverage nucleic acid molecule length may be between 50 and 500 bases.However, smaller nucleic acid molecules may be used in certainembodiments, particularly those where the nucleic acid is used as anantioxidant for an oil or other lipid-rich material in which it is notreadily soluble. Alternatively, larger nucleic acid molecules may beused in such materials to form small nucleic acid particles which may bedispersed throughout the material.

The compositions of the present invention may include nucleic acid asthe sole antioxidant, or they may also include other antioxidants,including both natural and synthetic antioxidants.

The following examples are provided only to illustrate certain aspectsof the invention and are not intended to embody the total scope of theinvention or the totality of any aspect thereof. Variations of theexemplary embodiments of the invention below will be apparent to oneskilled in the art and are intended to be included within the scope ofthe invention.

EXAMPLES Example 1 Nucleic Acid Stabilized Vitamin Compositions

Although Vitamin C is commonly sold as a nutrient supplement, because itis a potent antioxidant it has a very limited shelf-life. Furthermore,Vitamin C becomes a mild pro-oxidant after an oxidative reaction.Therefore, Vitamin C supplements that have been substantially oxidizedmay actually be harmful. Nucleic acid may be added to a solution ofVitamin C in an alcohol. The solution may be dried to produce Vitamin Cpowder or tablets. The optimal proportion of nucleic acid to Vitamin Cmay be determined by mixing various proportions in alcohol solutions,drying the solutions, and storing the dried powders for approximately 6months (the normal shelf-life of Vitamin C). Vitamin C levels orantioxidant activity may then be measured, for instance by measuringreactivity with diphenyl picryl hydrazine. In certain embodiments, theoptimal proportion of nucleic acid to Vitamin C by weight will fallbetween 60% and 80%. Examples of antioxidant formulations and methodsfor testing these formulations are provided in U.S. Pat. No. 6,235,721of Ghosal (the “'721 Patent”). Although the '721 Patent addressesstabilization of Vitamin C and other compounds using a fruit extract,the methodologies should be applicable to nucleic acid as well.

Nucleic acid may also be used as an antioxidant for Vitamin E. Methodssuch as those described in Example 5 may be used to increase solubilityof nucleic acid in Vitamin E. Alternatively, the nucleic acid may beallowed to from small aggregates in a mixture with the Vitamin E, or theVitamin E may be allowed to form small aggregates in a mixture with thenucleic acid. Proportions of nucleic acid used in Vitamin E formulationsmay be similar to those used in Vitamin C formulations, although morenucleic acid by weight may be recommended.

Nucleic acid may similarly be added to other vitamin compositions, whichmay or may not contain Vitamins C or E. In particular, Vitamin Asupplements may be prepared using methodologies similar to those usedfor Vitamin E.

Example 2 Nucleic Acid Stabilized Lotion Compositions

Lotions, creams and other similar preparations, whether used forcosmetic, topical pharmaceutical, or other purposes, are often formed bymixing component lipids and other chemicals at approximately 80–85° C.Lipids may oxidize during this heating process, so the addition of anantioxidant capable of enduring heating is desirable. Nucleic acid maybe added to such a mixture prior to or during heating to help preventoxidation of lipid or other lotion components. The amount of nucleicacid in such lotions may be varied depending upon the oxidativereactivity of the lotion components. For many lotions, between 0.1 and5% nucleic acid by weight may be sufficient. Appropriate nucleic acidproportions may be tested by allowing samples to sit for acceptableshelf-life time intervals, for example one year, and then measuring theamount of oxidative damage to one or more indicator compounds.

The above method may also be used to prepare lotions containingantioxidants, such as Vitamins C and E, or topical ointments which willcontain pharmaceuticals susceptible to oxidative damage.

Lotion, cream and topical antioxidant formulations may further beprepared as described in U.S. Pat. No. 6,124,268 (the “'268 Patent”).Although the '268 patent relates to the use of a fruit extract as anantioxidant, its methodologies should be applicable to nucleic acids aswell. Methods for increasing solubility of nucleic acids in hydrophobicsubstances are described in Example 5. Alternatively, nucleic acids maybe allowed to form small aggregates within the lotion.

Example 3 Nucleic Acid Stabilized Synthetic Estrogens

Synthetic estrogens are used for a variety of medical reasons, rangingfrom birth control to treatment of hormone deficiencies. However,synthetic estrogens tend to be unstable, especially in dampenvironments. One source of instability, hydrolysis of the syntheticmolecules, may be combated using a buffer. However, use of the bufferonly produces a shelf-life of around 6 months. Addition of 0.25 to 6moles of an antioxidant has been shown to increase shelf-life up to twoyears without significant degradation of alkali metal synthetic estrogensulfates. (See U.S. Pat. No. 4,154,820 of Simoons, the “'820 Patent”.)Nucleic acid may be used in as an antioxidant in synthetic estrogencompositions and tested for efficiency as described in the '820 Patent.Because both nucleic acid and most synthetic estrogens may besolubilized in water, aqueous solutions may be easily prepared and usedor further processed to gelcap or dry formulations.

Example 4 Nucleic Acid Stabilized Gum Base and High-Temperature Oils

Like lotions, gum base must often be heated during preparation. Thisallows significant oxidation of fats and oils in the gum base. Althoughgum base processing temperatures are often higher than the denaturationtemperature of DNA, they are not so high as to cause breakdown of thenucleic acid backbone. Many of DNA's antioxidant properties do notdepend upon a helical structure, therefore DNA should continue tofunction as an antioxidant even in denatured form. Additionally, the DNAmolecule may be modified by crosslinking to prevent denaturation athigher temperatures.

Addition of between 7–2000 parts per million of antioxidant has beenshown to significantly reduce oxidative damage to gum base, particularlyduring heat processing steps. (See U.S. Pat. No. 5,545,416 to Brodericket al., the “'416 Patent”.) Although the '416 Patent uses a syntheticantioxidant, its techniques should be applicable to the preparation of agum base with nucleic acid antioxidant. Additionally, as described inthe '416 Patent, larger amounts of nucleic acid may be added in initialprocessing stages to ensure that sufficient amounts remain after heatprocessing. This technique of adding larger amounts of nucleic acidprior to heat processing may also be used the processing of othermaterials.

This methodology may also be useful for the addition of nucleic acid asan antioxidant to oils used for frying, which may also involve the useof temperatures above the denaturation point of DNA. Another methodologyfor maintaining a constant nucleic acid concentration in cooking oilsthat may be used with nucleic acid as an antioxidant is described inU.S. Pat. No. 4,115,597 of Pellar.

Example 5 Solubilization of Nucleic Acid in Lipids

Nucleic acid is relatively insoluble in hydrophobic compositions, suchas fats, Vitamin E and other lipids. In order to increase itssolubility, it may be added to the hydrophobic composition in a mannersimilar to that described in U.S. Pat. No. 5,084,293 of Todd, Jr. (the“'293 Patent”) for ascorbic acid. Generally, the process in the '293Patent involves forming an anhydrous paste of the nucleic acid in asubstance that has both hydrophobic and hydrophilic properties.

Example 6 Nucleic Acid Aggregates

Nucleic acid may also be added to lipids by the incorporation of smallnucleic acid aggregates into the lipids. These aggregates are preferablyless than 50 microns in size. U.S. Pat. No. 5,296,249 of Todd, Jr. (the“'249 Patent”) discloses a method of producing and using Vitamin Cmicroparticles of approximately less than 38 microns. The '249 Patentshows that antioxidants do not have to be solubilized in a lipid toreduce oxidative damage. Small particles dispersed in the lipid are alsofunctional for this purpose. The wet milling techniques of the '249Patent may be used for nucleic acid in solution in which it is insolubleor only slightly soluble, such as a solution with high alcohol content.Alternatively, small nucleic acid particles may be produced by droppingaqueous nucleic acid solution into a condensing solution, such as a highalcohol content solution. Other methods of producing very smallparticles of nucleic acid are also possible. The size and type of thenucleic acid molecule may affect the size of the aggregate formed. Microor nanoparticles of nucleic acid may be preferred for many uses.

Example 7 Treatment of Fruits and Vegetables

Harvested fruits and vegetables deteriorate rapidly for a variety ofreasons, one of which is the oxidation of molecules in their surfacelayers. In order to prevent this oxidation, U.S. Pat. No. 6,403,139 toSardo et al. (the “'139 Patent”) discloses a method for applyingantioxidants to harvested fruits and vegetables. Similar applicationtechniques may be used to apply nucleic acid as an antioxidant. Thenucleic acid may be applied at a lower temperature than in the '139Patent if it is in an aqueous solution. Alternatively, if the nucleicacid is first mixed in a hydrophobic compound as described in Examples 5and 6, if the hydrophobic compound is solid at room temperature it mayremain on the fruit or vegetable more readily than nucleic acid alone,but will likely benefit from elevated application temperatures asdescribed in the '139 Patent.

Example 8 Nucleic Acid and Additional Antioxidant Compositions

U.S. Pat. No. 5,498,434 (the “'434 Patent”) and U.S. Pat. No. 5,427,814(the “'814 Patent”) describe beneficial effects of providing more thanone antioxidant in a given formulation. Specifically, both observe abeneficial effect when Vitamin E and lecithin are used together witheither Vitamin C or oil of rosemary. Nucleic acid may also be used insuch a mixture, with expected beneficial effects. Such formulation maybe prepared as described in the '814 and '434 Patents. Because nucleicacid is hydrophilic, the process described in the '814 Patent may bepreferred, with nucleic acid used in a manner similar to or in place ofVitamin C.

Example 9 Coupled Antioxidant Systems

U.S. Pat. No. 6,093,436 describes an antioxidant system in which anenzyme, glucose oxidase, serves as the primary antioxidant and aninorganic oxygen scavenger serves as a repository. The system istailored for a beverage system. Nucleic acid may be in place of aninorganic oxygen scavenger in systems such as these.

Example 10 Nucleic Acid Effervescent Antioxidant

U.S. Pat. No. 5,919,483 of Takaichi et al. (the “'483 Patent”) describesan effervescent composition containing an antioxidant. The compositionis water soluble and may be used to store antioxidant then allows itsready use in an aqueous solution. Compositions similar to thosedescribed in the '483 Patent may be prepared using nucleic acid as theantioxidant.

Example 11 Nucleic Acid Antioxidant for Rapid Degradation

In certain applications long-lasting antioxidants are not required ordesirable. For example, short-lived antioxidants may be useful inneutralizing the effects of chemical weapons such as mustard gas. Whensuch short-lived antioxidants are desirable then RNA or shorter nucleicacid molecules may be used.

Example 12 Other Antioxidant Uses

Nucleic acid antioxidants of the present invention may be used toneutralize any oxide, including peroxides, carbonyl radicals, carboxyradicals and ozones. They may also be used to mitigate effects ofbiological materials that directly or indirectly produce oxides such asprostaglandins and interleukins or other inflammatory mediators orby-products. Finally, oxidative products of any source such as UVradiation, mustard gas and alkylating agents may be neutralized or theireffects mitigated using antioxidants of the present invention. Forexample, nucleic acid antioxidants may mitigate effects of blisteringagents by neutralizing oxidative products and reducing inflammation.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of specific embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. All such similar substitutesand modifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention.

1. A method of preserving vitamins subject to oxidative damagecomprising adding a sufficient amount of purified nucleic acid to avitamin such that the rate of oxidative damage of the vitamin isreduced.
 2. The method of claim 1, wherein the nucleic acid comprisesDNA.
 3. The method of claim 1, wherein the vitamin comprises an aqueoussolution of hydrophilic vitamin subject to oxidative damage.
 4. Themethod of claim 1, wherein the purified nucleic acid is added in asolvent with both hydrophilic and hydrophobic regions, and the vitaminis a hydrophobic vitamin.
 5. The method of claim 1, wherein the nucleicacid is an aggregate of less than 50 μM in diameter, and the vitamin isa hydrophobic vitamin.
 6. The method of claim 1, wherein the purifiednucleic acid is sprayed onto the surface of the vitamin.
 7. A reducedoxidative vitamin composition comprising: a vitamin subject to oxidativedamage; and a sufficient amount of purified nucleic acid to reduce therate of oxidative damage to the vitamin, wherein the proportion ofnucleic acid to vitamin by weight is between 60% and 80%.
 8. Thecomposition method of claim 7, wherein the nucleic acid comprises DNA.